I have transfected a eukaryotic expression vector with desire gene in MDA MB cell line. when i was trying to do westernblot, we were getting band in positive control but not in the cell lysate and also the supernatant. Actually we are using DAB substrate for westernblot. what may be the reason for not getting band ?
Transfecting some extra cells with Green Fluorescent Protein (GFP) in parallel with your gene of interest would help you to estimate transfection efficiency you are getting even before you start any WB experiments. This will help you to exclude transfection issue and focus on other possible issues.
Also, do this cells express endogenous gene you are trying to express? If yes, endogenous protein can be a good control for the antibody you are using.
What is your positive control - purified recombinant proteing? And what is the supernatant: what step of what protocol produced it? How much of your positive control are you loading compared to cell lysate being loaded? Realize that even a transfected mammalian cell line won't have THAT much of the target protein compared to its total protein pool.
But assuming the major problem here is a lack of your target protein in the cell lysate, a few possibilities - 1) the transfection didn't work. Try using a different plasmid, or a different protein in the same plasmid, to see if you can detect expression from those. If a different plasmid works, your cells don't like the plasmid you gave them. If a different protein works, maybe the cells are just degrading your target protein.
2) Check your plasmid sequencing data - make sure you don't have an out-of-frame error or the like. Make sure that you don't have a mutation of the protein in the region being bound by your antibody.
3) Yes, DAB could be the problem. In general, colorimetic readouts for Western blots can be problematic at best. Try using a chemiluminescent approach - such reagents require a dark-room and film, or a dark box and camera, or whatnot, to work up, but also have much lower detection limits than any colorimetric approach is going to have.
Transfecting some extra cells with Green Fluorescent Protein (GFP) in parallel with your gene of interest would help you to estimate transfection efficiency you are getting even before you start any WB experiments. This will help you to exclude transfection issue and focus on other possible issues.
Also, do this cells express endogenous gene you are trying to express? If yes, endogenous protein can be a good control for the antibody you are using.
Dear Perumal,
You have already got wonderful suggestions. I would just like to add some more. It may be any of the following problems-
A. Transfection didn't work.
You can use a reporter plasmid to make sure your efficiency of transfection is good. If you have any other gene cloned in the same vector background and already standardized transfection conditions, you can use that also to check the transfection efficiency. You can use some other cell lines which are good for overexpression studies to see whether your construct has a cell specific expression problem or not.
B. Transfection worked but protein is unstable.
Your protein might not be stable inside the cell, or like william mentions it gets degraded. You can do certain things to be able to detect such problem-
a. Load very high amount of protein and see if at all any faint band is there.As mentioned check for endogenous band or if not then some other protein (some other gene cloned in same vector and overexpress it) to check whether its just your protein which is unstable or the cells tend to degrade any foreign protein.
b. You can prepare lysates at different time points.There will be a short window between the time when your protein gets expressed inside the cells and when degradation is at its peak. If you can detect it in that window then you still might be able to see it and know for sure that, indeed it is getting rejected by the host. Although it is recommended to prepare cell lysates after 48 hours, but you may try 24, 36 and then narrow it down to see where you get the best expression. Also if you know the subcellular localization of your protein, try to make that fraction and enrich it. For eg. if it is a nuclear protein , make nuclear lysate instead of whole cell extract to increase the abundance.
C. Transfection works, protein is expressed but cell lysis isn't proper.
Prepare lysate in some other buffer and also probe for some other protein (loading control) to check whether lysis is proper or not.
D. Transfection works, protein is expressed but gets degraded after lysis.
As mentioned already, add protease inhibitors, store lysates at -20 or 4 degree if you are regularly using. When you blot for your loading control, you can detect this problem, if other proteins are also getting degraded, then it can't be inside the cell, it has to be after lysis.
E. Lastly, Western blot didn't work.
But this might not be the reason in your case as you are getting the band for your positive control so you have the transfer conditions optimized.
You may try these to see where exactly is the problem. Hope it helps!
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